Catch and light up: Dual bioorthogonal reactions actualize fluorescent probe retention and activation for surgical navigation.

Fluorescent molecular probes are promising imaging tools for tumor surgical navigation. However, achieving both precise diagnosis and long-term surgical navigation in complex tumor environments remains challenging. In this study, we developed a "catch and light-up" fluorescent probe (CyTB) that enables specific fluorescence retention and activation, thereby enhancing imaging navigation accuracy. The probe consists of a hemicyanine unit as the near-infrared fluorescence signal reporter, tetrazine (Tz) reactive group bicyclo [6.1.0]nonyne (BCN) and trans-cyclooctene (TCO). Once arrival at the tumor site labeled by Tz in advance, the BCN group of probes was anchored through ligation reaction to enhance the probe retention. Concurrently, the TCO group can rapidly be cleaved by Tz leading to the light-up of fluorescence. The retention time of the probe at the tumor site can reach 130 h, while the tumor-to-normal tissue ratio in metastatic microtumor of pancreatic cancer (< 2 mm) still reaches 6.5, enabling efficient tumor tracking and precise marginal resection, thus providing a promising strategy for surgical navigation.